Recombinant Influenza Viruses for Vaccines and Gene Therapy

ABSTRACT

The invention provides compositions and methods useful to prepare segmented, negative strand RNA viruses, e.g., orthomyxoviruses such as influenza A viruses, entirely from cloned cDNAs and in the absence of helper virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.09/971,372, filed Oct. 4, 2001; which is a Continuation of InternationalPatent Application No. PCT/US00/09021, filed Apr. 5, 2000; which is anInternational filing of U.S. Provisional Application No. 60/127,912,filed Apr. 6, 1999, and U.S. Provisional Application No. 60/132,839,filed May 6, 1999; the disclosure of all of which are incorporatedherein by reference.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with a grant from the Government of the UnitedStates of America (grant AI-29599 from the National Institute of Allergyand Infectious Diseases Public Health Service). The Government may havecertain rights in the invention.

BACKGROUND OF THE INVENTION

The ability to generate infectious RNA viruses from cloned cDNAs hascontributed greatly to the biological understanding of these pathogensand hence to improved methods of disease control (Palese et al., 1996).However, this progress had been relatively limited for negative-sense ascompared with positive-sense RNA viruses, because neither the genomicviral RNA (vRNA) nor the antigenomic complementary RNA (cRNA) ofnegative-sense RNA viruses can serve as a direct template for proteinsynthesis. Rather, the vRNA, after its encapsidation by viralnucleoprotein (NP), must be transcribed into positive-sense mRNA by theviral RNA polymerase complex. Thus, the minimal replication unit isformed by the genomic vRNA complexed with NP and the polymeraseproteins. Despite these obstacles, reverse genetics methods have beenestablished to produce nonsegmented negative-sense RNA viruses,including rabies virus (Snell et al., 1994), vesicular stomatitis virus(Lawson et al., 1995); Whelan et al., 1995), measles virus (Radecke etal., 1995), respiratory syncytial virus (Collins et al., 1995), Sendaivirus (Garcin et al., 1995; Kato et al., 1996), rinderpest virus (Baronet al., 1997), human parainfluenza virus type 3 (Hoffman et al., 1997)and SV5 (He et al., 1997).

The Orthomyxoviridae, Arenaviridae, and Bunyaviridae families containsegmented, negative strand RNA genomes and include several human andanimal pathogens, for example, influenza virus types A, B, and C(Orthomyxoviridae), lymphocytic choriomeningitis virus (LCMV)(Arenaviridae), and encephalitic and hemorrhagic fever viruses(Bunyaviridae, Arenaviridae). Their genomes consist of two(Arenaviridae), three (Bunyaviridae), or six to eight (Orthomyxoviridae)single-stranded RNA molecules of negative polarity (complementary tomRNA). The vRNAs interact with NP and viral RNA-dependent RNA-polymeraseto form ribonucleoprotein complexes (RNPs). The RNPs are surrounded by alipid bilayer derived from the host cell. Inserted in this envelope areviral glycoproteins, which are essential for receptor binding and entryinto the host cell. Thus, generating segmented negative-sense RNAviruses from cloned cDNAs poses a formidable challenge, as one mustproduce a separate vRNA for each gene segment.

Bridgen and Elliott (1996) produced a Bunyamwera virus (familyBunyaviridae) from cloned cDNAs encoding three segments of antigenomic,positive-sense vRNA. However, the efficiency of virus recovery was low.None of the orthomyxoviruses, which contain six (thogotovirus), seven(influenza C virus) or eight (influenza A and B viruses) segments ofnegative-sense RNA have been produced entirely from cloned cDNAs. Thislag in progress has been felt most acutely in efforts to controlinfluenza virus infections.

Palese and colleagues (Enami et al., 1990) pioneered the reversegenetics, helper virus-dependent system for influenza A virus (FIG. 1A).In their approach, RNP complexes are generated by in vitro vRNAsynthesis in the presence of purified polymerase and NP proteins, andthen used to transfect eukaryotic cells. Subsequent infection withinfluenza A helper virus results in the generation of viruses possessinga gene derived from cloned cDNA. A second method, developed by Neumannet al. (1994), is based on the in vivo synthesis of vRNA by RNApolymerase I (FIG. 1B), a cellular enzyme that transcribes ribosomal RNAthat lacks both a 5′ cap and a 3′ polyA tail. Cells infected withinfluenza virus and transfected with a plasmid containing clonedinfluenza virus cDNA, flanked by murine RNA polymerase I promoter andterminator sequences, led to the production of transfectant viruses.With both methods, however, transfectants must be selected from a vastbackground of helper viruses, which requires a strong selection systemand complicates the generation of growth-defective viruses.

A system to generate replication-incompetent virus-like particles (VLPs)was developed by Mena et al. (1996), in which an influenza virus-likevRNA encoding a reporter gene is transcribed in vitro and transfectedinto eukaryotic cells. All ten influenza virus proteins are expressedfrom plasmids under the control of a T7 RNA polymerase promoter. Whenthe transfected cells are infected with a recombinant vaccinia virusthat expressed T7 RNA polymerase, they produced influenza VLPs. However,the efficiency of the system is low: in 25% of the experiments, theinvestigators failed to detect reporter gene expression. Moreover,vaccinia virus expresses more than 80 proteins, any of which couldaffect the influenza viral life cycle.

Thus, what is needed is a method to prepare segmented, negative strandRNA viruses, e.g., orthomyxoviruses such as influenza A viruses,entirely from cloned cDNAs.

SUMMARY OF THE INVENTION

The invention provides at least one of the following isolated andpurified vectors: a vector comprising a promoter operably linked to aninfluenza virus PA cDNA linked to a transcription termination sequence,a vector comprising a promoter operably linked to an influenza virus PB1cDNA linked to a transcription termination sequence, a vector comprisinga promoter operably linked to an influenza virus PB2 cDNA linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus HA cDNA linked to a transcriptiontermination sequence, a vector comprising a promoter operably linked toan influenza virus NP cDNA linked to a transcription terminationsequence, a vector comprising a promoter operably linked to an influenzavirus NA cDNA linked to a transcription termination sequence, a vectorcomprising a promoter operably linked to an influenza virus M cDNAlinked to a transcription termination sequence, and a vector comprisinga operably linked to an influenza virus NS cDNA linked to atranscription termination sequence. The cDNA may be in the sense orantisense orientation relative to the promoter. Thus, a vector of theinvention may encode an orthomyxovirus protein (sense), or vRNA(antisense). Any promoter may be employed to express a viral protein.Preferred promoters for the vectors encoding vRNA include, but are notlimited to, a RNA polymerase I promoter, a RNA polymerase II promoter, aRNA polymerase III promoter, a T7 promoter, and a T3 promoter. It isfurther preferred that the RNA polymerase I promoter is a human RNApolymerase I promoter. Preferred transcription termination sequences forthe vectors encoding vRNA include, but are not limited to, a RNApolymerase I transcription termination sequence, a RNA polymerase IItranscription termination sequence, or a RNA polymerase IIItranscription termination sequence, or a ribozyme. Preferably, thevectors comprise influenza cDNA, e.g., influenza A (e.g., any influenzaA gene including any of the 15 HA or 9 NA subtypes), B or C DNA (seeChapters 45 and 46 of Fields Virology (Fields et al. (eds.),Lippincott-Raven Publ., Philadelphia, Pa. (1996), which are specificallyincorporated by reference herein), although it is envisioned that thegene(s) of any virus may be employed in the vectors or methods of theinvention.

The invention provides a composition comprising a plurality of theorthomyxovirus vectors of the invention. In one embodiment of theinvention, the composition comprises: a) at least two vectors selectedfrom a vector comprising a promoter operably linked to an influenzavirus PA cDNA linked to a transcription termination sequence, a vectorcomprising a promoter operably linked to an influenza virus PB1 cDNAlinked to a transcription termination sequence, a vector comprising apromoter operably linked to an influenza virus PB2 cDNA linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus HA cDNA linked to a transcriptiontermination sequence, a vector comprising a promoter operably linked toan influenza virus NP cDNA linked to a transcription terminationsequence, a vector comprising a promoter operably linked to an influenzavirus NA cDNA linked to a transcription termination sequence, a vectorcomprising a promoter operably linked to an influenza virus M cDNAlinked to a transcription termination sequence, and a vector comprisinga operably linked to an influenza virus NS cDNA linked to atranscription termination sequence; and b) at least two vectors selectedfrom a vector encoding influenza virus PA, a vector encoding influenzavirus PB1, a vector encoding influenza virus PB2, and a vector encodinginfluenza virus NP. Preferably, the vectors encoding viral proteinsfurther comprise a transcription termination sequence. It is preferredthat a promoter for the vectors comprising influenza virus cDNA includesa RNA polymerase I promoter, a RNA polymerase II promoter, a RNApolymerase III promoter, a T7 promoter, and a T3 promoter. It is alsopreferred that each vector comprising influenza virus cDNA comprises atranscription termination sequence such as a RNA polymerase Itranscription termination sequence, a RNA polymerase II transcriptiontermination sequence, or a RNA polymerase III transcription terminationsequence, or a ribozyme. Preferably, the vectors comprise influenza DNA,e.g., influenza A, B or C DNA.

More preferably, the composition comprises a plurality of orthomyxovirusvectors, comprising: a) at least two vectors selected from a vectorcomprising a RNA polymerase I promoter operably linked to an influenzavirus PA cDNA linked to a RNA polymerase I transcription terminationsequence, a vector comprising a RNA polymerase I promoter operablylinked to an influenza virus PB1 cDNA linked to a RNA polymerase Itranscription termination sequence, a vector comprising a RNA polymeraseI promoter operably linked to an influenza virus PB2 cDNA linked to aRNA polymerase I transcription termination sequence, a vector comprisinga RNA polymerase I promoter operably linked to an influenza virus HAcDNA linked to a RNA polymerase I transcription termination sequence, avector comprising a RNA polymerase I promoter operably linked to aninfluenza virus NP cDNA linked to a RNA polymerase I transcriptiontermination sequence, a vector comprising a RNA polymerase I promoteroperably linked to an influenza virus NA cDNA linked to a RNA polymeraseI transcription termination sequence, a vector comprising a RNApolymerase I promoter operably linked to an influenza virus M cDNAlinked to a RNA polymerase I transcription termination sequence, and avector comprising a RNA polymerase I promoter operably linked to aninfluenza virus NS cDNA linked to a RNA polymerase I transcriptiontermination sequence; and b) at least two vectors selected from a vectorencoding influenza virus PA, a vector encoding influenza virus PB1, avector encoding influenza virus PB2, a vector encoding influenza virusNP, a vector encoding influenza virus HA, a vector encoding influenzavirus NA, a vector encoding influenza virus M1, a vector encodinginfluenza virus M2, and a vector encoding influenza virus NS2.

Another embodiment of the invention comprises a composition of theinvention as described above further comprising a vector comprising apromoter linked to 5′ orthomyxovirus non-coding sequences linked to adesired linked to 3′ orthomyxovirus non-coding sequences linked totranscription termination sequences. The introduction of such acomposition to a host cell permissive for orthomyxovirus replicationresults in recombinant virus comprising vRNA corresponding to sequencesof the vector comprising 5′ orthomyxovirus non-coding sequences linkedto a cDNA linked to 3′ orthomyxovirus non-coding sequences. Preferably,the cDNA is in an antisense orientation. Also preferably, the promoteris a RNA polymerase I promoter, a RNA polymerase II promoter, a RNApolymerase III promoter, a T7 promoter, and a T3 promoter. It is alsopreferred that the transcription termination sequence is a RNApolymerase I transcription termination sequence, a RNA polymerase IItranscription termination sequence, or a RNA polymerase IIItranscription termination sequence, or a ribozyme. For example, the cDNAmay encode an immunogenic epitope, such as an epitope useful in a cancertherapy or vaccine.

A plurality of the vectors of the invention may be physically linked oreach vector may be present on an individual plasmid or other, e.g.,linear, nucleic acid delivery vehicle.

The invention also provides a method to prepare influenza virus. Themethod comprises contacting a cell with a plurality of the vectors ofthe invention, e.g., sequentially or simultaneously, for example,employing a composition of the invention, in an amount effective toyield infectious influenza virus. The invention also includes isolatingvirus from a cell contacted with the composition. Thus, the inventionfurther provides isolated virus, as well as a host cell contacted withthe composition or isolated virus of the invention.

As described hereinbelow, influenza A viruses were prepared entirelyfrom cloned cDNAs. The reverse genetics approach described herein ishighly efficient and can be used to introduce mutations into any genesegment and to develop influenza virus-based gene delivery systems. Forexample, human embryonic kidney cells (293T) were transfected with eightplasmids, each encoding a viral RNA of the A/W5N/33 (H1N1) or A1PR18134(H1N1) virus, flanked by the human RNA polymerase I promoter and themouse RNA polymerase I terminator, together with plasmids encoding viralnucleoprotein and the PB2, PB1 and PA viral polymerases. This strategyyields >1×103 plaque-forming units (pfu) of virus per ml of supernatantat 48 hours post-transfection. Depending on the virus generated, theaddition of plasmids expressing all of the remaining viral structuralproteins led to a substantial increase in virus production, >3×10pfu/ml. Reverse genetics was also employed to generate a reassortantvirus containing the PB1 gene of the A/PR/8134 virus, with all othergenes representing A/WSN/33. Additional viruses produced by this methodhad mutations in the PA gene or possessed a foreign epitope in the headof the neuraminidase protein.

Moreover, the same approach may be employed for other viruses togenerate nonsegmented negative strand RNA viruses (i.e.,Paramyxoviridae, Rhabdoviridae, and Filoviridae), or other segmentednegative strand RNA viruses, e.g., Arenaviridae and Bunyaviridae,entirely from cloned cDNA. Further, the expression of cRNA in cellsinstead of vRNA may improve the efficiency of virus generation.

The method of the invention allows easy manipulation of influenzaviruses, e.g., by the introduction of attenuating mutations into theviral genome. Further, because influenza viruses induce strong humoraland cellular immunity, the invention greatly enhances these viruses asvaccine vectors, particularly in view of the availability of naturalvariants of the virus, which may be employed sequentially, allowingrepetitive use for gene therapy.

Thus, the invention provides isolated and purified vectors or plasmids,which express or encode influenza virus proteins, or express or encodeinfluenza vRNA, both native and recombinant vRNA. Thus, a vector orplasmid of the invention may comprise a gene or open reading frame ofinterest, e.g., a foreign gene encoding an immunogenic peptide orprotein useful as a vaccine. Preferably, the vector or plasmid whichexpresses influenza vRNA comprises a promoter, e.g., a RNA polymerase I,suitable for expression in a particular host cell, e.g., avian ormammalian host cells such as canine, feline, equine, bovine, ovine, orprimate cells including human cells. Also preferably, the vectors orplasmids comprising DNA useful to prepare influenza vRNA comprise RNApolymerase I transcription termination sequences. For vectors orplasmids comprising a gene or open reading frame of interest, it ispreferred that the gene or open reading frame is flanked by the 5′ and3′ non-coding sequences of influenza virus, and even more preferably,that the gene or open reading frame is operably linked to a RNApolymerase I promoter and RNA polymerase I transcription terminationsequence.

As described hereinbelow, 293T were transfected with plasmids encodingthe influenza A virus structural proteins, together with a plasznid thatcontained the green fluorescence protein (GFP) reporter gene, flanked byan RNA polymerase I promoter and terminator. Intracellular transcriptionof the latter construct by RNA polymerase I generated GFP vRNA that waspackaged into influenza virus-like particles. This system, whichproduced more than 10 infectious particles per ml of supernatant, may beuseful in studies of influenza virus replication and particle formation.It might also benefit efforts in vaccine production and in thedevelopment of improved gene therapy vectors.

Therefore, the invention also provides for a host cell, the genome ofwhich is stably augmented with at least one recombinant DNA molecule.The recombinant DNA molecule includes at least one of the following: arecombinant DNA molecule comprising a promoter functional in the hostcell linked to a first lox site linked to a DNA segment comprising atranscription stop or termination sequence linked to a second lox sitelinked to an influenza virus HA coding region; a recombinant DNAmolecule comprising a promoter functional in the host cell linked to afirst lox site linked to a DNA segment comprising a transcription stopor termination sequence linked to a second lox site linked to aninfluenza virus NA coding region; a recombinant DNA molecule comprisinga promoter functional in the host cell linked to a first lox site linkedto a DNA segment comprising a transcription stop or termination sequencelinked to a second lox site linked to an influenza virus M1 codingregion; a recombinant DNA molecule comprising a promoter functional inthe host cell linked to a first lox site linked to a DNA segmentcomprising a transcription stop or termination sequence linked to asecond lox site linked to an influenza virus NS2 coding region; arecombinant DNA molecule comprising a promoter functional in the hostcell linked to a first lox site linked to a DNA segment comprising atranscription stop or termination sequence linked to a second lox sitelinked to an influenza virus M2 coding region; a recombinant DNAmolecule comprising a promoter functional in the host cell linked to afirst loxP site linked to a DNA segment comprising a transcription stopor termination sequence linked to a second lox site linked to aninfluenza virus PA coding region; a recombinant DNA molecule comprisinga promoter functional in the host cell linked to a first lox site linkedto a DNA segment comprising a transcription stop or termination sequencelinked to a second lox site linked to an influenza virus PB1 codingregion; a recombinant DNA molecule comprising a promoter functional inthe host cell linked to a first lox site linked to a DNA segmentcomprising a transcription stop or termination sequence linked to asecond lox site linked to an influenza virus PB2 coding region; or arecombinant DNA molecule comprising a promoter functional in the hostcell linked to a first lox site linked to a DNA segment comprising atranscription stop or termination sequence linked to a second lox sitelinked to an influenza virus NP coding region.

Preferably, the host cell is augmented with a recombinant DNA moleculecomprising a promoter functional in the host cell linked to a first loxsite linked to a DNA segment comprising a transcription stop sequencelinked to a second lox site linked to an influenza virus HA codingregion; a recombinant DNA molecule comprising a promoter functional inthe host cell linked to a first lox site linked to a DNA segmentcomprising a transcription stop sequence linked to a second lox sitelinked to an influenza virus NA coding region; a recombinant DNAmolecule comprising a promoter functional in the host cell linked to afirst lox site linked to a DNA segment comprising a transcription stopsequence linked to a second lox site linked to an influenza virus M1coding region; a recombinant DNA molecule comprising a promoterfunctional in the host cell linked to a first lox site linked to a DNAsegment comprising a transcription stop sequence linked to a second loxsite linked to an influenza virus NS2 coding region; and a recombinantDNA molecule comprising a promoter functional in the host cell linked toa first lox site linked to a DNA segment comprising a transcription stopsequence linked to a second lox site linked to an influenza virus M2coding region. Preferably, the lox sites are loxP sites.

The invention also provides a method to prepare infectious replicationdefective influenza virus. The method comprises contacting a host cellwhich is augmented with at least one recombinant DNA molecule of theinvention, e.g., encoding HA, NA, M1, M2, NS2, PA, PB1, PB2, or NP, witha recombinant influenza virus comprising: vRNA comprising a Cre openreading frame, and vRNAs comprising influenza genes not expressed by thehost cell. Virus is then recovered from the contacted host cell.Preferably, the recombinant virus further comprises vRNA comprising adesired open reading frame. Alternatively, the augmented host cell iscontacted with a vector comprising a promoter functional in the hostcell operably linked to a DNA segment encoding Cre, and a plurality ofvectors each comprising a promoter operably linked to an influenza viruscDNA not present in the host cell. Virus is then recovered.

The invention also provides a host cell, the genome of which isaugmented with a recombinant DNA molecule comprising a promoterfunctional in the host cell linked to a first lox site linked to a DNAsegment comprising a transcription stop sequence linked to a second loxsite linked to a host cell surface binding protein coding region; arecombinant DNA molecule comprising a promoter functional in the hostcell linked to a first lox site linked to a DNA segment comprising atranscription stop sequence linked to a second lox site linked to afusion protein coding region; a recombinant DNA molecule comprising apromoter functional in the host cell linked to a first lox site linkedto a DNA segment comprising a transcription stop sequence linked to asecond lox site linked to an influenza virus M1 coding region; arecombinant DNA molecule comprising a promoter functional in the hostcell linked to a first lox site linked to a DNA segment comprising atranscription stop sequence linked to a second lox site linked to aninfluenza virus NS2 coding region; and a recombinant DNA moleculecomprising a promoter functional in the host cell linked to a first loxsite linked to a DNA segment comprising a transcription stop sequencelinked to a second lox site linked to an influenza virus M2 codingregion. Preferably, the lox sites are loxP sites.

Yet another embodiment is a host cell, the genome of which is augmentedwith a recombinant DNA molecule comprising a promoter functional in thehost cell linked to a first lox site linked to a DNA segment comprisinga transcription stop sequence linked to a second lox site linked to ahost cell surface binding and fusion protein coding region; arecombinant DNA molecule comprising a promoter functional in the hostcell linked to a first lox site linked to a DNA segment comprising atranscription stop sequence linked to a second lox site linked to aninfluenza virus M1 coding region; a recombinant DNA molecule comprisinga promoter functional in the host cell linked to a first lox site linkedto a DNA segment comprising a transcription stop sequence linked to asecond lox site linked to an influenza virus NS2 coding region; and arecombinant DNA molecule comprising a promoter functional in the hostcell linked to a first lox site linked to a DNA segment comprising atranscription stop sequence linked to a second lox site linked to aninfluenza virus M2 coding region. Preferably, the lox sites are loxPsites.

Host cells augmented with recombinant DNA molecules as describedhereinabove are useful to prepare infectious replication defectiveinfluenza virus. For example, a host cell stably transformed withrecombinant DNA molecules encoding HA, NA, M1, M2 and NS2 is contactedwith a plurality of vectors, i.e., vectors which express vRNA comprisinga Cre open reading frame, vRNA comprising PA, vRNA comprising NP, vRNAcomprising PB1, vRNA comprising PB2, and optionally, vRNA comprising agene of interest; and vectors which encode PA, PB1, PB2, and NP.

The methods of producing virus described herein, which do not requirehelper virus infection, are useful in viral mutagenesis studies, and inthe production of vaccines (e.g., for AIDS, influenza, hepatitis B,hepatitis C, rhinovirus, filoviruses, malaria, herpes, and foot andmouth disease) and gene therapy vectors (e.g., for cancer, AIDS,adenosine deaminase, muscular dystrophy, ornithine transcarbamyIasedeficiency and central nervous system tumors).

Thus, a virus for use in medical therapy (e.g., for a vaccine or genetherapy) is provided. For example, the invention provides a method toimmunize an individual against a pathogen, e.g., a bacteria; virus, orparasite, or a malignant tumor. The method comprises administering tothe individual an amount of at least one isolated virus of theinvention, optionally in combination with an adjuvant, effective toimmunize the individual. The virus comprises vRNA comprising apolypeptide encoded by the pathogen or a tumor specific polypeptide.

Also provided is a method to augment or increase the expression of anendogenous protein in a mammal having an indication or diseasecharacterized by a decreased amount or a lack of the endogenous protein.The method comprises administering to the mammal an amount of anisolated virus of the invention effective to augment or increase theamount of the endogenous protein in the mammal. Preferably, the mammalis a human.

The invention also provides vectors and methods for the recombinantproduction of positive strand viruses, e.g., positive-sense RNA viruses.Thus, the invention provides a vector comprising a DNA segmentcomprising RNA polymerase I transcription initiation sequences operablylinked to a second DNA segment comprising sequences from apositive-sense RNA virus, optionally operably linked to a third DNAsegment comprising RNA polymerase I transcription termination sequences.Also provided is a method of using the vector(s) to prepare recombinantvirus. The method is particularly useful as it employs cloned DNA andtransfection techniques, thus circumventing RNA handling. Moreover, RNApolymerase I transcription is highly efficient and has high fidelity.For positive-sense RNA viruses whose genomic RNA is uncapped (e.g.,pestiviruses; hepatitis C virus; and Picornaviridae, includingpoliovirus, rhinoviruses, hepatitis A virus, and foot and mouth diseasevirus), a cDNA encoding the full-length genome is introduced ingenomic-sense orientation between RNA polymerase I promoter andterminator sequences. Transfection of the resulting plasmid intopermissive host cells yields genomic RNA for virus replication. A numberof positive-sense RNA viruses contain capped genomic RNAs (e.g.,flaviviruses, including dengue fever virus and several encephalitisviruses). While RNA polymerase I transcripts are not capped, a cDNAencoding the full-length genome of RNA viruses having capped genomicRNAs is introduced in antigenomic-sense orientation in a RNA polymeraseI transcription vector. Following transfection of the resulting plasmid,cellular RNA polymerase I transcribes an antigenomic (uncapped) RNA.Moreover, cotransfection with protein expression plasmids for theproteins required for replication results in the replication of theantigenomic RNA, hence yielding genomic RNA and ultimately infectiousvirus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of established reverse genetics systems. Inthe RNP transfection method (A), purified NP and polymerase proteins areassembled into RNPs with use of in vitro-synthesized vRNA. Cells aretransfected with RNPs, followed by helper virus infection. In the RNApolymerase I method (B), a plasmid containing the RNA polymerase Ipromoter, a cDNA encoding the vRNA to be rescued, and the RNA polymeraseI terminator is transfected into cells. Intracellular transcription byRNA polymerase I yields synthetic vRNA, which is packaged into progenyvirus particles upon infection with helper virus. With both methods,transfectant viruses (i.e., those containing RNA derived from clonedcDNA), are selected from the helper virus population.

FIG. 2. Schematic diagram of the generation of RNA polymerase Iconstructs. cDNAs derived from influenza virus were amplified by PCR,digested with BsmBI and cloned into the BsmBI sites of the pHH21 vector(E. Hoffmann, Ph.D. thesis, Justus, Liebig-University, Giessen,Germany), which contains the human RNA polymerase I promoter (P) and themouse RNA polymerase I terminator (T). The thymidine nucleotide upstreamof the terminator sequence (*T) represents the 3′ end of the influenzaviral RNA Influenza A virus sequences are shown in bold face letters.The numeric numbers in parentheses positioned next to the nucleotidesequences represent the SEQ ID NOs for the sequences.

FIG. 3. Proposed reverse genetics method for generating segmentednegative-sense RNA viruses. Plasmids containing the RNA polymerase Ipromoter a cDNA for each of the eight viral RNA segments, and the RNApolymerase I terminator are transfected into cells together with proteinexpression plasmids. Although infectious viruses can be generated withplasmids expressing PA, PB1, PB2, and NP, expression of all remainingstructural proteins (shown in brackets) increases the efficiency ofvirus production depending on the virus generated.

FIG. 4. Detection of the FLAG epitope in cells infected with atransfectant virus. Antibody staining was used to identify the NA inMDCK cells infected with either PR8-WSN-FL79 (A, D) or A/WSN/33wild-type virus (B, E), or on mock-infected MDCK cells (C, F). Ninehours after infection, cells were fixed with paraformaldehyde, treatedwith Triton X-100 and incubated with either anti-FLAG (A-C) or anti-WSNNA (D-F) monoclonal antibodies. Intensive Golgi staining (red) isapparent in positive samples (A, D, and E).

FIG. 5. Recovery of PA mutants. The PA gene of each virus was amplifiedby RT-PCR with primers that yield a 1226 bp fragment (position 677 to1903 of the mRNA, lanes 1, 3, 5), which was then digested with therestriction enzyme Bsp1201 (at position 846 of the rriRNA, lanes 4, 7)or Pvull (at position 1284 of the mRNA, lanes 2, 6). The presence ofBsp1201 or Pvull sites in the PCR products yielded either 169 bp and1057 bp or 607 bp and 619 bp fragments, respectively. MW=molecularweight markers.

FIGS. 6A and 6B. Primers employed to amplify influenza sequences. TheSEQ ID NO for each primer is presented as a numeric number in aparentheses positioned next to the primer.

FIG. 7. The pPolI-GFP plasmid for generating influenza virus-like RNAencoding the GFP protein. This plasmid contains the GFP gene (derivedfrom pEGFP-N1; Clontech, Palo Alto, Calif.) in antisense orientationbetween the 5′ and 3′ noncoding regions of influenza A virus segment 5,flanked by the human RNA polymerase I promoter and the mouse RNApolymerase I terminator. The numeric number in a parenthesis positionednext to a nucleotide sequence represents the SEQ ID NO for thatsequence.

FIG. 8. Schematic diagram of VLP generation strategy. Individual proteinexpression plasmids and a plasmid containing the RNA polymerase Ipromoter, a cDNA encoding the GFP reporter gene, and the RNA polymeraseI terminator are transfected into 293T cells. Intracellulartranscription by RNA polymerase I yields GFP vRNA of negative polarity,as indicated by inverted letters. Supernatants containing VLPs areharvested, mixed with influenza helper virus and inoculated into MDCKcells.

FIG. 9. The PA, PB1, PB2, and NP proteins of influenza A virusencapsidate GFP vRNA produced by RNA polymerase I, leading to GFPexpression. 293T cells were transfected with plasmids expressing thePB2, PB1, PA and NP proteins (A) or with all plasmids except the oneexpressing the NP protein (B), together with the RNA polymerase 1-GFPgene plasmid for intracellular synthesis of reporter gene vRNA. Cellswere fixed 48 h after transfection, and GFP expression was determinedwith a fluorescence microscope.

FIG. 10. Generation of infectious influenza VLPs. 293T cells weretransfected with nine plasmids, each expressing a different viralstructural protein (A), or with eight plasmids omitting the constructfor NP (B), together with the RNA polymerase I-GFP gene plasmid.Forty-eight hours after transfection, VLP-containing supernatants werecollected, mixed with A/WSN/33 helper virus, and inoculated into MDCKcells. Cells were fixed at 10 hours after infection, and GFP expressionwas determined with a fluorescence microscope.

FIG. 11. Schematic of the use of Cre recombinase to express influenzaNS2 protein in a cell, the genome of which is augmented with arecombinant DNA molecule. The genome of the cell comprises a recombinantDNA molecule which comprises a promoter linked to a site specificrecombination site (e.g., loxP) linked to a transcription stop sequencelinked to a second site specific recombination site in the sameorientation as the first site specific recombination site linked to theNS2 gene.

FIG. 12. Preparation of replication defective influenza virus.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “isolated and/or purified” refer to in vitropreparation, isolation and/or purification of a vector or plasmid of theinvention, so that it is not associated with in vivo substances, or issubstantially purified from in vitro substances. As used herein, theterm “recombinant nucleic acid” or “recombinant DNA sequence or segment”refers to a nucleic acid, e.g., to DNA, that has been derived orisolated from a source, that may be subsequently chemically altered invitro, so that its sequence is not naturally occurring, or correspondsto naturally occurring sequences that are not positioned as they wouldbe positioned in the native genome. An example of DNA “derived” from asource, would be a DNA sequence that is identified as a useful fragment,and which is then chemically synthesized in essentially pure form. Anexample of such DNA “isolated” from a source would be a useful DNAsequence that is excised or removed from said source by chemical means,e.g., by the use of restriction endonucleases, so that it can be furthermanipulated, e.g., amplified, for use in the invention, by themethodology of genetic engineering.

As used herein, “site-specific recombination” is intended to include thefollowing three events: 1) deletion of a target DNA segment flanked bysite-specific recombination sites or sequences, e.g., loxP sites; 2)inversion of the nucleotide sequence of a target DNA segment flanked bysite-specific recombination sites or sequences, e.g., lox sites; and 3)reciprocal exchange of target DNA segments proximate to site-specificrecombination sites or sequences, e.g., lox sites located on differentDNA molecules. Site-specific recombinase systems include, but are notlimited to, the Cre/loxP system of bacteriophage P1 (U.S. Pat. No.5,658,772).

To remedy the reversibility of a site-specific recombination reaction,the structure of the recombination system may be altered. Thesite-specific recombination sequence can be mutated in a manner that theproduct of the recombination reaction is no longer recognized as asubstrate for the reverse reaction, thereby stabilizing the integrationor excision event. For example, to remove undesired sequences, lox sitesin the same orientation are positioned to flank the undesired sequences.

Other lox sites include loxB, loxL, and loxR sites which are nucleotidesequences isolated from E. coli (Hoess et al., Proc. Natl. Acad. Sci.USA, 79 3398 (1982)). Lox sites can also be produced by a variety ofsynthetic techniques which are known in the art. For example, synthetictechniques for producing lox sites are disclosed by Ito et al., Nuc.Acid Res., 10, 1755 (1982) and Ogilvie et al., Science, 214, 270 (1981).

As used herein, the expression “lox site” means a nucleotide sequence atwhich the gene product of the cre gene can catalyze a site-specificrecombination. LoxP is a 34 base pair nucleotide sequence which can beisolated from bacteriophage P1 by methods known in the art (see, forexample, Hoess et al., Proc. Natl. Acad. Sci. USA, 79, 3398 (1982)).LoxP consists of two 13 base pair inverted repeats separated by an 8base pair spacer region.

As used herein, the expression “cre gene” means a nucleotide sequencewhich codes for an enzymic gene product which effects site-specificrecombination of DNA in eukaryotic cells at lox sites. A cre gene can beisolated from bacteriophage P1 by methods known in the art (see Abremaidet al., Cell, 32, 1301-1311 (1983)).

Influenza Virus Replication

Influenza A viruses possess a genome of eight single-strandednegative-sense viral RNAs (vRNAs) that encode a total of ten proteins.The influenza virus life cycle begins with binding of the hemagglutinin(HA) to sialic acid-containing receptors on the surface of the hostcell, followed by receptor-mediated endocytosis. The low pH in lateendosomes triggers a conformational shift in the HA, thereby exposingthe N-terminus of the HA2 subunit (the so-called fusion peptide). Thefusion peptide initiates the fusion of the viral and endosomal membrane,and the matrix protein (M1) and RNP complexes are released into thecytoplasm. RNPs consist of the nucleoprotein (NP), which encapsidatesvRNA, and the viral polymerase complex, which is formed by the PA, PB1,and PB2 proteins. RNPs are transported into the nucleus, wheretranscription and replication take place. The RNA polymerase complexcatalyzes three different reactions: synthesis of an mRNA with a 5′ capand 3′ polyA structure, of a full-length complementary RNA (cRNA), andof genomic vRNA using the cDNA as a template. Newly synthesized vRNAs,NP, and polymerase proteins are then assembled into RNPs, exported fromthe nucleus, and transported to the plasma membrane, where budding ofprogeny virus particles occurs. The neuraminidase (NA) protein plays acrucial role late in infection by removing sialic acid fromsialyloligosaccharides, thus releasing newly assembled virions from thecell surface and preventing the self aggregation of virus particles.Although virus assembly involves protein-protein and protein-vRNAinteractions, the nature of these interactions is largely unknown.

Thogotovirus

Thogotoviruses (THOV) represent a new genus in the family ofOrthomyxoviridae. They are transmitted by ticks and have been found indomestic animals, including camels, goats, and cattle. Consequently,THOV can replicate in tick and vertebrate cells. The THOV genomecomprises six segments of single-stranded, negative-sense RNA. Theproteins encoded by the three largest segments show significant homologyto the influenza virus polymerase proteins PB2, PB1, and PA. Segment 5encodes a protein related to influenza virus NP. The THOV glycoprotein,which is encoded by segment 4, is not homologous to either influenzavirus HA or NA, but it shows sequence similarity to the Baculovirusglycoprotein. The smallest segment is thought to encode a matrix proteinand does not resemble any of the influenza virus proteins. Likeinfluenza virus, both the 3′ and 5′ ends of the vRNA are required forpromoter activity, and this activity is located in the terminal 14 and15 nucleotides of the 3′ and 5′ ends of the vRNA, respectively.

The mRNA synthesis of THOV is primed by host cell-derived capstructures. However, in contrast to influenza virus, only the capstructures (without additional nucleotides) are cleaved from cellularmRNAs (Albo et al., 1996; Leahy et al., 1997; Weber et al., 1996). Invitro cleavage assays revealed that both the 5′ and 3′ ends of vRNA arerequired for endonuclease activity (Leahy et al., 1998), but addition ofa model cRNA promoter does not stimulate endonuclease activity (Leahy etal., 1998), as has been shown for influenza virus (Clanci et al., 1995;Hagen et al., 1994). A ‘hook’ structure has been proposed for THOV(Leahy et al., 1997; Weber et al., 1997), which is similar to thecorkscrew structure proposed for influenza virus (Flick et al., 1996).This ‘hook’ structure, however, is only found in the THOV vRNA promoter.The cRNA promoter sequence does not allow the formation of base pairsbetween positions 2 and 9, and between 3 and 8 at the 5′ end of thecRNA. Alterations at positions 3 or 8 to allow base-pairing betweenthese nucleotides stimulates endonuclease activity, which is strongsupporting evidence of the proposed ‘hook’ structure (Leahy et al.,1998). Moreover, this structure might be crucial for the regulation ofthe THOV life cycle; the vRNA promoter, forming the ‘hook’ structure,may stimulate PB2 endonuclease activity, thereby allowing transcription.The cRNA promoter, in contrast, may not form the ‘hook’ structure andmay therefore be unable to stimulate endonuclease activity, thusresulting in replication.

Bunyaviridae

The family Bunyaviridae includes several viruses that cause hemorrhagicor encephalitic fevers in humans (e.g., Rift fever valley, Hantaan, LaCrosse, and Crimean-Congo hemorrhagic fever). The spherical andenveloped virions contain three segments of single-stranded,negative-sense RNA (reviewed in Elliott, 1997). The largest segment (L)encodes the viral RNA polymerase protein (L protein), whereas the Msegment encodes the two viral glycoproteins G1 and G2, and anonstructural protein (NSm). The smallest segment (S) encodes thenucleocapsid protein (N) and a second nonstructural protein (NSs). Virusreplication and transcription take place in the cytoplasm, and newlyassembled virions bud through the membranes of the Golgi apparatus.

Bridgen & Elliott (1996) have established a reverse genetics system togenerate infectious Bunyamwera virus entirely from cloned cDNAs. Theyfollowed a strategy first described by Schnell et al. (1994) for rabiesvirus: intracellular transcription of a cDNA coding for thepositive-sense antigenomic RNA (but not for the negative-sense genomicRNA) in cells expressing the viral polymerase and nucleoprotein. Bridgen& Elliott (1996) infected HeLaT4+ cells with vaccinia virus expressingT7 polymerase and transfected these cells with plasmids expressingproteins encoded by the S, M, and L segments. They then transfectedthese cells with three plasmids encoding full-length anti-genomic cDNAsflanked by the T7 polymerase promoter and the hepatitis delta virusribozyme. To increase the number of bunyavirus particles relative to thenumber of vaccinia virus particles, the authors used mosquito cells inwhich Bunyamwera but not Vaccinia virus replicates. This protocol can beused not only to genetically engineer Bunyaviridae, but also generatereassortant viruses that cannot easily be obtained by coinfecting cellswith different Bunyaviridae strains.

To study bunyavirus promoter elements and the viral proteins that arerequired for transcription and replication, Dunn et al. (1995) clonedthe CAT gene in the negative-sense orientation between the 5′ and 3′nontranslated regions of the Bunyamwera S RNA segment. Cells weretransfected with constructs expressing the proteins encoded by the L andS segment and were then transfected with in vitro transcribed RNA, whichresulted in CAT activity. The bunyavirus S segment encodes two proteins,N and NSs, in overlapping reading frames. To determine whether both ofthese proteins are required for transcription and replication,constructs expressing only N or NSs were tested for CAT activity. Nprotein expression, together with L protein, resulted in CAT activity,whereas no CAT activity was detected with the NSs expression construct.Thus, the L and N proteins are sufficient for transcription andreplication of a bunyavirus-like RNA.

As with influenza virus, the terminal sequences of bunyavirus RNAs arecomplementary and highly conserved. It has therefore been assumed thatthese sequence elements define the bunyaviral promoter and are crucialfor promoter activity. Deletion of five nucleotides at the 3′ end of theviral RNA drastically reduces CAT expression (Dunn et al., 1995). Incontrast, addition of two nucleotides at the 5′ end, or of 11 or 35nucleotides at the 3′ end does not abolish CAT expression (Dunn et al,1995). Therefore, like the influenza virus polymerase complex, thebunyavirus polymerase protein can apparently start transcription and/orreplication internally.

The invention will be further described by the following examples.

Example 1 Materials and Methods

Cells and viruses. 293T human embryonic kidney cells and Madin-Darbycanine kidney cells (MDCK) were maintained in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% fetal calf serum and in modifiedEagle's medium (MEM) containing 5% newborn calf serum, respectively. Allcells were maintained at 37° C. in 5% CO2. Influenza viruses A/WSN/33(H1N1) and A/PR/8/34 (H1N1) were propagated in 10-day-old eggs.

Construction of plasmids. To generate RNA polymerase I constructs,cloned cDNAs derived from A/WSN/33 or A/PR/8/34 viral RNA wereintroduced between the promoter and terminator sequences of RNApolymerase I. Briefly, the cloned cDNAs were amplified by PCR withprimers containing BsmBI sites, digested with BsmBI, and cloned into theBsmBI sites of the pHH21 vector which contains the human RNA polymeraseI promoter and the mouse RNA polymerase I terminator, separated by BsmBIsites (FIG. 2). The PB2, PB1, PA, HA, NP, NA, M, and NS genes of theA/WSN/33 strain were PCR-amplifled by use of the following plasmids:pSCWPB2, pGW-PB1, and pSCWPA (all obtained from Dr. Debi Nayak at theUniversity of California Los Angeles), and pWH17, pWNP152, pT3WNA15(Castrucci et al., 1992), pGT3WM, and pWNS1, respectively. The PB1 geneof influenza A/PR/8/34 virus was amplified by using pcDNA774 (PB1)(Perez et al., 1998) as a template. See FIG. 6 for the sequences of theprimers. To ensure that the genes were free of unwanted mutations,PCR-derived fragments were sequences with an autosequencer (AppliedBiosystem Inc., CA, USA) according to the protocol recommended by themanufacturer. The cDNAs encoding the HA, NP, NA, and M1 genes ofAAVSN/33 virus were cloned as described (Huddleston et al., 1982) andsubcloned into the eukaryotic expression vector pCAGGS/MCS (controlledby the chicken 13-actin promoter) (Niwa et al., 1991), resulting inpEWSN-HA, pCAGGS-WSN-NP0-14, pCAGGS-WNA15, and pCAGGS-WSN-M1-2/1,respectively. The M2 and NS2 genes from the A/PR/8/34 virus wereamplified by PCR and cloned into pCAGGS/MCS, yielding pEP24c andpCA-N52. Finally, pcDNA774 (PB1), pcDNA762 (PB2), and pcDNA787 (PA) wereused to express the PB2, PB1, and PA proteins under control of thecytomegalovirus promoter (Perez et al., 1998).

Generation of infectious influenza particles. 293T cells (1×106) weretransfected with a maximum of 17 plasmids in different amounts with useof Trans IT LT-1 (Panvera, Madison, Wis.) according to themanufacturer's instructions. Briefly, DNA and transfection reagent weremixed (2 pl Trans IT-LT-1 per jig of DNA), incubated at room temperaturefor 45 minutes and added to the cells. Six hours later, theDNA-transfection reagent mixture was replaced by Opti-MEM (Gibco/BRL,Gaithersburg, Md.) containing 0.3% bovine serum albumin and 0.01% fetalcalf serum. At different times after transfection, viruses wereharvested from the supernatant and titrated on MDCK cells. Since helpervirus was not required by this procedure, the recovered transfectantviruses were analyzed without plaque purification.

Determination of the percentage of plasmid-transfected cells producingviruses. Twenty-four hours after transfection, 293T cells were dispersedwith 0.02% EDTA into single cells. The cell suspension was then diluted10-fold and transferred to confluent monolayers of MDCK cells in 24-wellplates. Viruses were detected by the hemagglutination assay.

Immunostaining assay. Nine hours after infection with influenza virus,cells were washed twice with phosphate-buffered saline (PBS) and fixedwith 3.7% paraformaldehyde (in PBS) for 20 minutes at room temperature.Next, they were treated with 0.1% Triton X-100 and processed asdescribed by Neumann et al. (1997).

Results

Generation of infectious virus by plasmid-driven expression of viral RNAsegments, three polymerase subunits and NP protein. Althoughtransfection of cells with a mixture of RNPs extracted from purifiedvirions results in infectious influenza particles, this strategy is notlikely to be efficient when used with eight different in vitro generatedRNPs. To produce infectious influenza viruses entirely from cDNAs, eightviral RNPs were generated in vivo. Thus, plasmids were prepared thatcontain cDNAs for the full-length viral RNAs of the A/′WSN/33 virus,flanked by the human RNA polymerase I promoter and the mouse RNApolymerase I terminator. In principle, transfection of these eightplasmids into eukaryotic cells should result in the synthesis of alleight influenza vRNAs. The PB2, PB1, PA and NP proteins, generated bycotransfection of protein expression plasmids, should then assemble thevRNAs into functional vRNPs that are replicated and transcribed,ultimately forming infectious influenza viruses (FIG. 3). 1×10 293Tcells were transfected with protein expression plasmids (1 pg ofpcDNA762 (PB2), 1 pg of cDNA774 (PB1), 0.1 gg of pcDNA787 (PA), and 1 pgof pCAGGS-WSN-NP0/14) and 1 pg of each of the following RNA polymerase Iplasmids (pPolI-WSN-PB2, pPolI-WSN-PB1, pPolI-WSN-PA, pPolI-WSN-HA,pPolI-WSN-NP, pPolI-WSN-NA, pPolI-WSN-M, and pPolI-WSN-NS). The decisionto use a reduced amount of pcDNA787 (PA) was based on previousobservations (Mena et al., 1996), and data on the optimal conditions forgeneration of virus-like particles (VLPs) (data not shown). Twenty-fourhours after transfection of 293T cells, 7×103 phi of virus per ml wasfound in the supernatant (Experiment 1, Table 1), demonstrating for thefirst time the capacity of reverse genetics to produce influenza A virusentirely from plasmids.

TABLE 1 Plasmid sets used to produce influenza virus from cloned cDNA*Experiment 1 2 3 4 5 6 7 8 RNA polymerase I plasmids for: † PB1 + + − −− − − − PR8- − − + + + + + + PB1 PB2 + + + + + + + + PA + + + + + + + +HA + + + + + + + + NP + + + + + + + + NA + + + + + + + +M + + + + + + + + NS + + + + + + + + Protein expression plasmids for:PB1 + + + + − + + + PB2 + + + + + − + + PA + + + + + − +NP + + + + + + + − HA − + − + + + + + NA − + − + + + + + M1 − +− + + + + + M2 − + − + + + + + NS2 − + − + + + + + Virus titer 7 × 10³ 7× 10³ 1 × 10³ 3 × 10⁴ 0 0 0 0 (pfu/ml) *293T cells were transfected withthe indicated plasmids. Twenty-four (Experiments 1 and 2) or forty-eighthours (Experiments 3-8) later, the virus titer in the supernatant wasdetermined in MDCK cells. † Unless otherwise indicated, plasmids wereconstructed with cDNAs representing the RNAs of AJWSN/33 virus.

Efficiency of influenza virus production with coexpression of all viralstructural proteins. Although expression of the viral NP and polymeraseproteins is sufficient for the plasmid-driven generation of influenzaviruses, it was possible that the efficiency could be improved. Inprevious studies, the expression of all influenza virus structuralproteins (PB2, PB1, PA, HA, NP, NA, M1, M2, and NS2) resulted in VLPsthat contained an artificial vRNA encoding a reporterchloramphenicol-acetyltransferase gene (Mena et al., 1996). Thus, theavailability of the entire complement of structural proteins, instead ofonly those required for viral RNA replication and transcription, mightimprove the efficiency of virus production. To this end, 293T cells weretransfected with optimal amounts of viral protein expression plasmids(as judged by VLP production; unpublished data): 1 ug of pcDNA762 (PB2)and pcDNA774 (PB1); 0.1 μg of pcDNA787 (PA); 1 μg of pEWSN-HA,pCAGGS-WSN-NP0/14, and pCAGGS-WNA15; 2 μg of pCAGGS-WSN-M1-2/1; 0.3 μgof pCA-NS2; and 0.03 μg of pEP24c (for M2), together with 1 jig of eachRNA polymerase I plasmid (Experiment 2, Table 1). A second set of cellswas transfected with the same set of RNA polymerase I plasmids, with theexception of the PB1 gene, for which pPolI-PR/8/34-PB1 was substitutedin an effort to generate a reassortant virus, together with plasmidsexpressing only PA, PB1, PB2, and NP (Experiment 3, Table 1) or thoseexpressing all the influenza structural proteins (Experiment 4, Table1). Yields of WSN virus did not appreciably differ at 24 hours(Experiments 1 and 2, Table 1) or at 36 hours (data not shown)post-transfection. However, more than a 10-fold increase in yields ofthe virus with PR/8/34-PB1 was found when all the influenza viralstructural proteins were provided (Experiments 3 and 4, Table 1).Negative controls, which lacked one of the plasmids for the expressionof PA, PB1, PB2, of NP proteins, did not yield any virus (Experiments5-8, Table 1). Thus, depending on the virus generated, expression of allinfluenza A virus structural proteins appreciably improved theefficiency of the reverse genetics method.

Next, the kinetics of virus production after transfection of cells wasdetermined using the set of plasmids used to generate a virus with theA/PR/8/34-PB1 gene. In two of three experiments, virus was firstdetected at 24 hours after transfection. The titer measured at thattime, >103 pfu/ml, had increased to >106 pfu/ml by 48 hours aftertransfection (Table 2). To estimate the percentage ofplasmid-transfected cells that were producing viruses, 293T cells weretreated with EDTA (0.02%) at 24 hours after transfection to disperse thecells, and then performed limiting dilution studies. In this experiment,no free virus was found in the culture supernatant at this time point.The results indicated that 1 in 103-3 cells was generating infectiousvirus particles.

TABLE 2 Kinetics of virus production after plasmid transfection into293T cells* Virus titers in culture supernatant (pfu/ml) Hours afterplasmid Experiment transfection 1 2 3 6 0 ND ND 12 0 ND 0 18 0 ND 0 24 0 2 × 10³ 6 × 10³ 30 ND  5 × 10⁴ 9 × 10⁴ 36 6 × 10² >1 × 10⁵ 7 × 10⁵ 42ND >1 × 10⁶ 5 × 10⁶ 48 8 × 10⁴ >1 × 10⁶ 1 × 10⁷ *293T cells weretransfected with eight RNA polymerase I plasmids encoding A/WSN/33 virusgenes with the exception of PB1 gene, which is derived from AJPR/8/34virus, and nine protein expression plasmids as described in the text. Atdifferent time points, we titrated virus in the culture supernatant inMDCK cells. ND = not done.

Recovery of influenza virus containing the FLAG epitope in the NAprotein. To verify that the new reverse genetics system allowed theintroduction of mutations into the genome of influenza A viruses, avirus containing a FLAG epitope (Castrucci et al., 1992) in the NAprotein was generated. 293T cells were transfected with an RNApolymerase I plasmid (pPolI-WSN-NA/FL79) that contained a cDNA encodingboth the NA protein and a FLAG epitope at the bottom of the protein'shead, together with the required RNA polymerase I and protein expressionplasmids. To confirm that the recovered virus (PR8-WSN-FL79) did in factexpress the NA-FLAG protein, immunostaining assays of cells infectedwith PR8-WSN-FL79 or A/WSN/33 wild-type virus was performed. Amonoclonal antibody to the FLAG epitope detected cells infected withPR8-WSN-FL79, but not those infected with wild-type virus (FIG. 4).Recovery of the PR8-WSN-FL79 virus was as efficient as that for theuntagged wild-type virus (data not shown). These results indicate thatthe new reverse genetics system allows one to introduce mutations intothe influenza A virus genome.

Generation of infectious influenza virus containing mutations in the PAgene. To produce viruses possessing mutations in the PA gene, two silentmutations were introduced creating new recognition sequences forrestriction endonucleases (Bspl 201 at position 846 and Pvull atposition 1284 of the mRNA). Previously, it was not possible to modifythis gene by reverse genetics, because of the lack of a reliableselection system Transfectant viruses, PA-T846C and PA-A1284 wererecovered. The recovered transfectant viruses were biologically clonedby two consecutive limiting dilutions. To verify that the recoveredviruses were indeed transfectants with mutations in the PA gene, cDNAfor the PA gene was obtained by reverse transcriptase-PCR. As shown inFIG. 5, PA-T846C and PA-A1284C viruses had the expected mutations withinthe PA gene, as demonstrated by the presence of the newly introducedrestriction sites. PCR of the same viral samples and primers without thereverse transcription step failed to produce any products (data notshown), indicating that the PA cDNA was indeed originated from vRNAinstead of the plasmid used to generate the viruses. These resultsillustrate how viruses with mutated genes can be produced and recoveredwithout the use of helper viruses.

Discussion

The reverse genetics systems described herein allows one to efficientlyproduce influenza A viruses entirely from cloned cDNAs. Bridgen andElliott (1996) also used reverse genetics to generate a Bunyamwera virus(Bunyaviridae family), but it contains only three segments ofnegative-sense RNA, and the efficiency of its production was low, 102pfu/107 cells. Although the virus yields differed among the experiments,consistently >103 pfu/106 cells was observed for influenza virus, whichcontains eight segments. There are several explanations for the highefficiency of the reverse genetics system described hereinabove. Insteadof producing RNPs in vitro (Luytjes et al., 1989), RNPs were generatedin vivo through intracellular synthesis of vRNAs using RNA polymerase Iand through plasmid-driven expression of the viral polymerase proteinsand NP. Also, the use of 293T cells, which are readily transfected withplasmids (Goto et al., 1997), ensured that a large population of cellsreceived all of the plasmids needed for virus production. In addition,the large number of transcripts produced by RNA polymerase I, which isamong the most abundantly expressed enzymes in growing cells, likelycontributed to the overall efficiency of the system. These features ledto a correspondingly abundant number of vRNA transcripts and adequateamounts of viral protein for encapsidation of vRNA, formation of RNPs inthe nucleus, and export of these complexes to the cell membrane, wherenew viruses are assembled and released.

Previously established reverse genetics systems (Enami et al., 1990;Neumann et al., 1994; Luytjes et al., 1989; Pleschka et al., 1996)require helper-virus infection and therefore selection methods thatpermit a small number of transfectants to be retrieved from a vastnumber of helper viruses. Such strategies have been employed to generateinfluenza viruses that possess one of the following cDNA-derived genes:PB2 (Subbarao et al., 1993), HA (Enami et al., 1991: Horimoto et al.,1994), NP (Li et al., 1995), NA (Enami et al., 1990), M (Castrucci etal., 1995; Yasuda et al., 1994), and NS (Enami et al., 1991). Most ofthe selection methods, except for those applicable to the HA and NAgenes, rely on growth temperature, host range restriction, or drugsensitivity, thus limiting the utility of reverse genetics forfunctional analysis of the gene products. Even with the HA and NA genes,for which reliable antibody-driven selection systems are available, itis difficult to produce viruses with prominent growth defects. Incontrast, the reverse genetics system described herein does not requirehelper virus and permits one to generate transfectants with mutations inany gene segment or with severe growth defects. This advantage isdemonstrated in FIG. 5, which the recovery of transfectant viruses witha mutated PA gene. Having the technology to introduce any viablemutation into the influenza A virus genome will enable investigators toaddress a number of long-standing issues, such as the nature ofregulatory sequences in nontranslated regions of the viral genome,structure-function relationships of viral proteins, and the molecularbasis of host-range restriction and viral pathogenicity.

Although inactivated influenza vaccines are available, their efficacy issuboptimal due partly to their limited ability to elicit local IgA andcytotoxic T cell responses. Clinical trials of cold-adapted liveinfluenza vaccines now underway suggest that such vaccines are optimallyattenuated, so that they will not cause influenza symptoms, but willstill induce protective immunity (reviewed in Keitel & Piedra, 1998).However, preliminary results indicate that these live virus vaccineswill not be significantly more effective than the best inactivatedvaccine (reviewed in Keitel. & Piedra, 1998), leaving room for furtherimprovement. One possibility would be to modify a cold-adapted vaccinewith the reverse genetics system described above. Alternatively, onecould start from scratch by using reverse genetics to produce a “master”influenza A strain with multiple attenuating mutations in the genes thatencode internal proteins. The most intriguing application of the reversegenetics system described herein may lie in the rapid production ofattenuated live-virus vaccines in cases of suspected pandemics involvingnew HA or NA subtypes of influenza virus.

This new reverse genetics system will likely enhance the use ofinfluenza viruses as vaccine vectors. The viruses can be engineered toexpress foreign proteins or immunogenic epitopes in addition to theinfluenza viral proteins. One could, for example, generate viruses withforeign proteins as a ninth segment (Enami et al., 1991) and use them aslive vaccines. Not only do influenza viruses stimulate strongcell-mediated and humoral immune responses, but they also afford a widearray of virion surface HA and NA proteins (e.g., 15 HA and 9 NAsubtypes and their epidemic variants), allowing repeated immunization ofthe same target population.

Influenza VLPs possessing an artificial vRNA encoding a reporter genehave been produced by expressing viral structural proteins and vRNA withthe vaccinia-T7 polymerase system (Mena et al., 1996). Using reversegenetics, one can now generate VLPs containing vRNAs that encodeproteins required for vRNA transcription and replication (i.e., PA, PB1,PB2, and NP), as well as vRNAs encoding proteins of interest. Such VLPscould be useful gene delivery vehicles. Importantly, their lack of genesencoding viral structural proteins would ensure that infectious viruseswill not be produced after VLP-gene therapy. Since the influenza virusgenome is not integrated into host chromosome, the VLP system would besuitable for gene therapy in situations requiring only short-termtransduction of cells (e.g., for cancer treatment). In contrast toadenovirus vectors (Kovesdi et al., 1997), influenza VLPs could containboth HA and NA variants, allowing repeated treatment of targetpopulations.

The family Orthomyxoviridae comprises influenza A, B, and C viruses, aswell as the recently classified Thogotovirus. The strategy forgenerating infectious influenza A viruses entirely from cloned cDNAsdescribed herein would apply to any orthomyxovirus, and perhaps to othersegmented negative-sense RNA viruses as well (e.g., Bunyaviridae,Arenaviridae). The ability to manipulate the viral genome withouttechnical limitations has profound implications for the study of virallife cycles and their regulation, the function of viral proteins and themolecular mechanisms of viral pathogenicity.

Example 2

Expression of the influenza virus proteins PB2, PB1, PA, and NP leads toreplication and transcription of an artificial viral RNA. To generateinfluenza VLPs, the RNA polymerase I system for the intracellularsynthesis of influenza viral RNAs in vivo was employed (FIG. 7). In thissystem, a cDNA encoding a reporter gene in antisense orientation isflanked by the 5′ and 3′ noncoding regions of an influenza viral RNA.This cassette is inserted between an RNA polymerase I promoter andterminator. Transfection of such constructs into eukaryotic cells leadsto transcription of the reporter gene by cellular RNA polymerase I,thereby generating influenza virus-like RNAs (Neumann et al., 1994).Upon influenza virus infection, the artificial vRNAs are replicated andtranscribed by the viral polymerase complex, resulting in the expressionof the reporter gene.

To determine whether expression of the PB2, PB1, PA, and NP proteinsleads to expression of the reporter gene encoded by the RNA polymeraseI-derived transcript, plasmids (liig each) expressing the NP protein ofA/WSN/33 (H1N1) virus under control of the chicken f3-actin promoter(pCAGGS-WSN-NP0/14), the polymerase proteins of A/PR/8/34 virus undercontrol of the cytomegalovirus promoter [pcDNA762 (PB2), pcDNA774 (PB1),and pcDNA787 (PA)], and an RNA polymerase I reporter gene construct(pPolI-GFP) were transfected into human embryonic kidney (293T) cells.Forty eight hours later, 30%-40% of the cells were expressing GFP (FIG.9). In contrast, GFP expression could not be detected in transfectedcells lacking the polymerase or NP proteins. These results indicatedthat NP and the three influenza viral polymerase proteins had formed afunctional complex that replicated and transcribed the RNA polymeraseI-derived GFP vRNA.

Optimal vRNA transcription and replication. To determine the amounts ofplasmid DNA required for optimal reporter GFP expression, we modulatedthe expression of the polymerase proteins and NP. Previous studies hadindicated that large amounts of PA reduce the extent of reporter geneexpression in transcription/replication systems (Mena et al., 1996).Therefore, in a stepwise manner, the expression of PA from the plasmidwas reduced, identifying 0.1 μg of pcDNA787 (PA) as the template amountyielding the strongest expression of GFP. With NP, the major structuralcomponent of RNP complexes, high amounts of protein expression plasmidmay be required. However, higher amounts of the plasmid did notappreciably affect the number of GFP-positive 293T cells. In addition,various amounts of the PB2 and PB1 protein expression plasmids (rangingfrom 1.0 to 0.03 μg) did not affect the GFP expression in 293T cells.Hence, in all subsequent experiments, 0.1 μg of pcDNA787 (PA), and 1.0μg of pcDNA774 (PB1), pcDNA762 (PB2), and pCAGGS-WSN-NP0/14, was used.

Formation of influenza VLPs from cloned cDNAs. Previous studies with thevaccinia virus T7 RNA polymerase system showed that the formation ofinfluenza VLPs requires nine influenza virus proteins: PB2, PB1, PA, HA,NA, NP, M1, M2, and NS2 (Mena et al., 1996). The NS1 protein, bycontrast, is dispensable for particle formation (Mena et al., 1996). Toestablish an efficient plasmid-driven system for VLP generation, cDNAswere generated that encoded the HA, NA, M1, M2, and NS2 genes. The cDNAswere cloned into the eukaryotic expression vector pCAGGS/MCS (controlledby the chicken E1-actin promoter), resulting in pEWSN-HA, pCAGGS-WNA15,pCAGGS-WSN-M1-2/1, pEP24c, and pCA-NS2, respectively. Expression of eachprotein was confirmed by Western blot analysis.

To generate VLPs, 106 293T cells were transfected with 1.0 μg of eachprotein expression plasmids (with the exception of pcDNA787 (PA), forwhich 0.1 μg was employed), and with 1 μg of the reporter gene constructpPolI-GFP. Culture supernatants were harvested 48 hours aftertransfection and mixed with A/WSN/33 virus to provide the influenzavirus proteins required for replication and transcription of GFP vRNA.The mixture was then inoculated into MDCK cells. Ten hours afterincubation, GFP-positive MDCK cells were detected, corresponding to 450particles/ml of supernatant (Table 3). Thus, plasmid-driven expressionof all influenza viral structural proteins resulted in the formation ofinfectious influenza VLPs containing GFP vRNA. Moreover, GFP vRNA wasdelivered to MDCK cells.

Optimal assembly of influenza virus. VLP formation was also studied incells expressing different amounts of the RNA polymerase I reporter geneconstruct, as well as HA, NA, M1, M2, and NS2 plasmid DNAs. Inexperiments with pPolI-GFP, 1.0 μg of the plasmid DNA was highlyefficient in generating VLPs, whereas the efficiency was significantlyreduced for 2.0 μg or 3.0 μg. Because the NS2 and M2 proteins areexpressed in low amounts late in infection, it was likely thatrelatively small amounts of the expression plasmids would be needed foroptimal VLP formation. Reduction of the M2 expression construct from 1.0μg to 0.3 μg resulted in a more than tenfold increase in the number ofGFP-positive MDCK cells (Table 3). Further reduction of plasmid to 0.03μg did not increase the number of VLPs. For NS2, lower amounts ofplasmid tested (0.1 μg) were associated with less efficient formation ofVLPs (Table3).

The M1 protein is the major structural component of the virion. Thus,high levels of M1 expression are likely required for efficient formationof VLPs. This prediction was tested in experiments comparing VLPformation in cells transfected with 1.0 μg or 2.0 μg of M1 plasmid DNA.As shown in Table 3, higher amounts of plasmid resulted in a more thantenfold increase in the number of GFP-positive MDCK cells. Comparison oftwo different amounts (1 g vs. 2 μg) of plasmids expressing the HA andNA proteins did not reveal any appreciable differences in VLP formation,leading to selection of 1 μg of each plasmid (pEWSN-HA, pCAGGS-WNA15)for use in subsequent experiments. Overall, these studies resulted ina >100-fold increase in the efficiency of VLP formation, ultimatelyleading to the production of more than 104 infectious influenza virusparticles per ml of supernatant (FIG. 10).

TABLE 3 Optimal amounts of plasmid DNA for the formation of infectiousVLPs.* Amount (μg) of plasmid DNA expressing: GFP Relative EfficiencyPB2 PB1 PA HA NP NA MI M2 NS2 vRNA of VLP formation^(†) 1.0 1.0 0.1 1.01.0 1.0 1.0 1.0 1.0 1.0 1 1.0 1.0 0.1 1.0 1.0 1.0 1.0 0.1 1.0 1.0 28 1.01.0 0.1 1.0 1.0 1.0 1.0 0.03 1.0 1.0 17 1.0 1.0 0.1 1.0 1.0 1.0 1.0 0.11.0 1.0 28 1.0 1.0 0.1 1.0 1.0 1.0 1.0 0.1 0.3 1.0 24 1.0 1.0 0.1 1.01.0 1.0 1.0 0.1 0.1 1.0 11 1.0 1.0 0.1 1.0 1.0 1.0 1.0 0.1 1.0 1.0 281.0 1.0 0.1 1.0 1.0 1.0 2.0 0.1 1.0 1.0 220 *293T cells were transfectedwith expression plasmids for all nine influenza virus structuralproteins and with the RNA polymerase I-GFP gene plasmid. Forty-eighthours after transfection, VLP-containing supernatants were collected,mixed with AJWSN/33 helper virus, and inoculated into MDCK cells. Thecells were fixed 10 h after infection and GFP expression was determinedwith a fluorescence microscope. Only the amounts of the Ml, M2, and NS2plasmids were varied (bold letter) to determine their optimal amountsfor GFP expression in MDCK cells. f The relative efficiency of VLPformation was determined by counting the number of GFP-positive cells infive microscopic fields. The sample containing 1 μg of each plasmid(which yielded 450 infectious VLP/ml of supernatant) was chosen as thereference (value of 1).

Authenticity of VLPs produced entirely from plasmids. To verify thatVLPs initiate infection in the same manner as authentic influenzaviruses, VLPs were neutralized with antibody to the WSN HA.VPL-containing supernatants derived from plasmid-transfected 293T cellswere incubated with a pool of anti-WSN HA monoclonal antibodies or witha monoclonal antibody to the G protein of vesicular stomatitis virus(VSV) (negative control) for 1 hour at room temperature. A/PR/8/34helper virus, which is not neutralized by the pool of anti-WSN HAmonoclonal antibodies, was added to the mixture and inoculated into MDCKcells. Only the anti-WSN-HA-specific monoclonal antibody neutralized theVLPs, indicating that the HA medicates the attachment and entry of VLPsinto cells.

Next, the minimal set of proteins required for the formation of VLPs wasidentified. Other have established that the three influenza viruspolymerases and the NP are essential for the replication andtranscription of yRNA (Honda et al., 1988). Therefore, each of thesefour proteins was included, but HA, NA, M1, M2, or NS2 was consecutivelyomitted. Exclusion of any of these plasmids did not affect thereplication/transcription of GFP vRNA in transfected 293T cells.Supernatants derived from transfected 293T cells that lacked the HA, NA,M1, or NS2 protein did not promote GFP expression in infected MDCKcells, indicating the absence of infectious VLPs. Infectious VLPs weredetected with omission of M2 but the number was low (>500 fold reductioncompared to the full set of structural proteins). Thus, all influenzavirus structural proteins are required for the efficient formation ofinfectious VLPs, in accord with data from studies of the vaccinia-virussystem (Mena et al., 1996).

VSV glycoprotein can replace the HA and NA proteins in the production ofVLPs. The influenza virus HA and NA proteins were replaced with the VSVG protein, which functions in receptor binding and fusion. In 293T cellstransfected with pPolI-GFP; optimal amounts of the PB2, PB1, PA, NP, M1,M2, and NS2 expression constructs; and 1 ug of the VSV-G construct(pCAGGS-VSV-G), substitution of the VSV-G protein for influenza virusglycoproteins did not adversely affect VLP formation. To the contrary,higher numbers of GFP-positive cells were reproducibly found when VSV-G,rather than the HA and NA, served as the viral glycoprotein. Thus, theVSV G protein can be efficiently incorporated into influenza virions andcan function as well as the HA and NA in virus release and entry.

An efficient system for generating infectious influenza virus particleswould be an asset in research with this virus and potentially in theproduction of vaccines and vectors for gene therapy. In contrast to theextant vaccinia virus system, the VLP production strategy described hereis highly efficient, both in the initial transfection of cells and inthe yield of VLPs (>104 infectious particles/ml of supernatant).Moreover, it is driven entirely by plasmids expressing influenza virusproteins (i.e., in the absence of any other viral proteins), whichgreatly simplifies the interpretation of results. Another majoradvantage is the capability to study the effects of lethal mutations invirion formation, packaging of RNP complexes, budding of virusreplication, and binding and fusion processes. In addition, it is likelythat the system described hereinabove would operate equally well withother viruses, e.g., paramyxoviruses and rhabdoviruses.

Influenza virus HA and NA proteins can be functionally replaced by theVSV glycoprotein G. Previously, it had been reported that influenzaviruses failed to incorporate VSV G protein when provided by recombinantSV40 virus (Nairn et al., 1993). The results described herein suggestthat neither the HA nor the NA is essential for the formation of VLPs,although it cannot be ruled out that these glycoproteins play a role ininteractions with other viral proteins, thus affecting the structure ofvirions, as suggested by the elongated shapes of viruses expressingtail-less HAs, NAs, or both (Garcia-Sastre et al., 1995; Jin et al.,1994; Jin et al., 1997; Mitnaul et al., 1996).

The plasmid-based system described hereinabove may be particularlyuseful for therapeutic gene delivery. VLPs can be prepared that containthe vRNA encoding the proteins required for transcription andreplication (i.e., the NP and the polymerases), as well as a vRNAencoding the protein of interest. These particles are infectious and candeliver a designated gene into target cells, where it would replicateand be transcribed. Because these particles do not contain a completecomplement of viral genes, they can not produce infectious progenyviruses. This feature, together with the lack of integration of theviral genome into host chromosomes, would ensure the biological safetyof gene delivery in human and nonhuman subjects. Finally, theavailability of 15 HA and 9 NA subtypes and their variants would allowthe repeated administration of VLPs, thereby overcoming immunoresistanceto vector-generated proteins, one of the major obstacles faced withrepeated use of other viral vectors, such as adenoviruses. A furtherbenefit of the plasmid-driven system would be realized in situationsrequiring only short-term expression of foreign proteins, as in cancertreatment.

Example 3

By using the Cre-loxP system, one can generate packaging cell lines forthe production of replication-defective viruses. For example, a proteinexpression vector is prepared that contains a transcription stopcassette (e.g., pBS302 of Life Technologies, Bethesda, Md.; and Sauer etal., 1993; Lasko et al., 1992; Pichel et al., 1993; Bolivar et al.,1977; Stuhl et al., 1981; Stuhl, 1985; Fiers et al., 1978), flanked bytwo loxP sites, and one of the viral genes. Transcription, initiated atthe promoter sequence, is blocked at the transcription stop sites. Thus,the viral gene is not transcribed and translated. A cell that is stablytransfected with such a vector is infected with an influenza virus thatlacks the vRNA encoding the gene cloned into the loxP system. This virusalso contains an additional vRNA encoding the Cre protein. This virus isnot viable in normal cells, because it lacks one of its vRNAs. However,in the packaging cell line, the Cre protein which is expressed from thevRNA results in recombination at the loxP site, resulting in thedeletion of the transcription stop site. Thus, the respective viralgene(s) is now transcribed and expressed, allowing the virus to amplifyin these cells (FIG. 11).

In addition, packaging cell lines are prepared that express the lateviral proteins (i.e., HA, NA, M1, M2, and NS2) controlled by the loxPsystem (FIG. 12). The HA and NA can be replaced by other viralreceptor-binding and fusion proteins (e.g., Ebola GP, Marburg GP,Bunyaviridae glycoproteins GP1 and GP2, the G and/or F proteins ofrhabdovirus and paramyxovirus, thogotovirus glycoprotein, and theglycoproteins of positive-strand RNA viruses). Virus-like particles aregenerated which contain the vRNAs encoding the proteins required forreplication/transcription (i.e., the polymerase and NP proteins), a vRNAencoding the gene of interest, and a vRNA encoding Cre. These vRNAs arepackaged into virus-like particles in the packaging cell lines.

These virus-like particles can be used for vaccine and gene therapypurposes because (i) they do not contain the full complement of viralgenes and thus no infectious progeny particles can be formed, meetingthe stringent safety concerns; (ii) they will likely express the foreignprotein at high levels; (iii) they do not express the viralglycoproteins (HA, NA) that are the major antigens; thus, the hostimmune response against the viral' proteins should be limited.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1-100. (canceled)
 101. A method for producing influenza virus particlesfor preparation of a vaccine, comprising: growing reassortant influenzavirus to produce virus particles and recovering influenza virusparticles produced by said growing for preparation of a vaccine, saidreassortant influenza virus being a recovered reassortant influenzavirus that was produced in cultured cells by introducing into culturedcells expression vectors which direct the expression of the genomic vRNAsegments or antigenomic cRNA segments of said genomic vRNA segments,said cells providing a nucleoprotein, and an RNA dependent RNApolymerase so that ribonucleoprotein complexes containing the genomicvRNA segments of said reassortant influenza virus can be formed andwherein said reassortant influenza virus can be assembled within saidcells in the absence of helper virus and wherein said cells werecultured so that said reassortant influenza virus was produced.
 102. Themethod of claim 101, wherein the cultured cells are from a cell linewhich comprises expression vectors which direct expression of saidnucleoprotein and the subunits of said RNA-dependent RNA polymerase.103. The method of claim 101, wherein said virus is an influenza virusof type A, B or C.
 104. The method of claim 101, wherein saidreassortant influenza virus contains vRNA segments derived from morethan one parent virus.
 105. The method of claim 101, wherein saidexpression vectors were capable of directly expressing genomic vRNAsegments of said virus.
 106. The method of claim 101, wherein saidgrowing of reassortant influenza virus to produce virus particles occursin an egg.
 107. The method of claim 101 further comprising attenuatingsaid virus particles.
 108. The method of claim 101, wherein all of saidexpression vectors are introduced by transfection into said cells by useof a liposomal transfection reagent.
 109. The method of claim 101,wherein said expression vectors were all plasmids.
 110. The method ofclaim 101, wherein each genomic vRNA segment of said virus or thecorresponding antigenomic cRNAs is encoded on a separate expressionvector.
 111. The method of claim 101, wherein the expression of eachgenomic vRNA segment or antigenomic cRNA is under the control of apromoter sequence which is a mammalian RNA polymerase I promoter. 112.The method of claim 111, wherein said mammalian RNA polymerase Ipromoter sequence is a human RNA polymerase I promoter.
 113. The methodof claim 101, wherein a coding sequence for each genomic vRNA segment orantigenomic cRNA in said expression vectors is upstream of transcriptionterminator.
 114. The method of claim
 102. wherein said component was aviral nucleoprotein.
 115. The method of claim 114, wherein said cell isa 293 T cell.
 116. A method for producing an influenza virus comprising:(a) producing in a cell all of the genomic vRNA of an influenza virus byintroducing into the cell DNA encoding at least one of said genomic vRNAor antigenomic cRNA corresponding to said genomic vRNA of the virus, (b)producing in the cell RNA dependent polymerase and nucleoprotein andassembling in the cell the influenza virus, said cell being free ofhelper virus.